This invention relates generally to novel compounds which inhibit the serotonin N-acetyltransferase enzyme, modulate the serotonin-melatonin pathway and various methods of use for the compounds of the invention.
The enzyme serotonin N-acetyltransferase, is responsible for the daily rhythmic cycle of melatonin in animals and man. Hormonal influences over circadian rhythm are currently poorly understood. While a wide array of hormones are known to cycle throughout the day, their precise roles in the physiology of the sleep/wake cycle have remained elusive. The hormone melatonin (5-methoxy-N-acetyltryptamine) is hypothesized to play an important role in the regulation of circadian rhythm. Discovered in 1917 as an extract of pineal gland which could lighten frog skin (1), melatonin's structure was not elucidated until 1959 by Aaron Lerner (2,3). Subsequently, its serum levels were found to cycle with diurnal periodicity, rising 10-100 times its daytime levels at night (FIG. 1) (4). What melatonin is really doing and how it is doing in both animals and man remain mysterious (5).
The circadian clocks in living organisms are thought to regulate basal body temperature, locomotor activity, sleep, eating behavior, and hormone production (5). In non-mammalian vertebrates, the pineal gland is the principal regulator of the circadian clock (6). In mammals including humans, the brain's suprachiasmatic nucleus (SCN) is believed to contain the "master clock" with outputs to and inputs from the pineal gland (5-7). The current model is that the suprachiasmatic nucleus sends outputs via the noradrenergic pathway to regulate pineal gland activity and the SCN contains melatonin receptors which allow for pineal gland input (FIG. 2) (5).
Serotonin is the most abundant hormone produced in the pineal, and the pineal is its site of highest concentration in the brain (5,6). Serotonin is a hormone-neurotransmitter with hypothesized roles in physiologic processes such as sleep and in pathophysiologic conditions including depression, chronic pain, and migraine, and drug addiction (8). Three major classes of clinical anti-depressant agents are thought to achieve their effect by stimulating the serotonin pathway, including monoamine oxidase inhibitors (e.g. deprenyl), tricyclic antidepressants (e.g. imipramine), and serotonin re-uptake inhibitors (e.g. prozac) (8). It is not currently known to what extent pineal-derived serotonin contributes to serotonin's overall physiological effects in man.
Serotonin is a biosynthetic precursor of melatonin and the pineal is the principal source of melatonin in the body (5,6). Despite significant investigation, melatonin's physiologic roles are not yet well understood. Melatonin's primary function is suggested to be entrainment of circadian rhythm in response to changes in external light (5). In this role, melatonin would be important in temperature regulation, mood, and the sleep-wake cycle (5, 9, 10). Solid evidence has also been obtained for melatonin's role in reproductive fitness in mammals (11, 12). More speculative claims have been advanced concerning melatonin's possible roles in effecting the immune, cardiovascular, and gastro-intestinal systems and in playing a role in cancer, aging, anorexia, and psychiatric disorders (Table 1). There is relatively little hard experimental evidence to validate or refute these claims at present (25).
Studies involving pinealectomy and those involving exogenous melatonin administration to discern melatonin's function have been reasonably compelling in confirming melatonin's role in circadian rhythm regulation in mammals. Pinealectomized rats maintained in constant light show a major disruption in wheel-running activity compared to controls (26). Exogenous melatonin administration has been shown to cause phase shifting in rat activity as well as in humans (27, 28). An intact SCN appears necessary for melatonin's entrainment effects.
In keeping with melatonin's role as a central nervous system hormone, receptor binding sites have been reported in discrete regions of the mammalian brain. These binding sites were classified as G-protein coupled receptors on the basis of pertussis-toxin sensitive adenylyl cyclase inhibition and affinity modulation by guanine nucleotides (29). In the last 3-4 years, two mammalian melatonin receptors have been identified by expression cloning and shown to have expression patterns consistent with their predicted locations from hormone binding studies (30, 31). High concentrations of receptor nRNAs have been detected in the suprachiasmatic nucleus of the hypothalamus and the hypophyseal pars tuberalis.
In addition, there are recent reports of intracellular receptors for melatonin. These receptors are "orphan" members of the steroid hormone/Zn-finer DNA binding family (32, 33). The K.sub.d of melatonin binding and activation appears to be considerably higher, 10-100 fold compared to that of the G-protein family, and their physiologic relevance is still unclear.
Melatonin is routinely purchased at over the counter drug and health-food stores and used by many individuals to attempt to relieve jet lag, insommia, loss of vitality with aging, problems with shift work, and affective disorders. The oral bioavailability in people is reportedly quite variable (25-fold range, refs. 5, 34). Even with the lower doses that are sold (5-10 mg), this can drive serum levels hundreds of times higher than physiologic night-time serum levels (ca 500 pM) Consequently, effects that are observed may be due to alternative signalling pathways than the normal endogenous melatonin-responsive pathways. While there are currently no reported toxic effects of pharmacologic melatonin dosing (300 mg/d, the melatonin content of about 1.5 million pineal glands is approved for clinical trials), the long-term effects of massive doses of melatonin are relatively unexplored (5).
The biosynthesis of melatonin begins with L-tryptophan (Table 2). This naturally occurring amino acid is present in the diet and uptake by the brain is dependent on specific transport mechanisms. The first step in melatonin biosynthesis is hydroxylation of the tryptophan indole ring at the 5-position to afford 5-hydroxytryptophan. This step is rate-limiting in serotonin production and is catalyzed by the mitochondrial enzyme non-heme iron monooxygenase, tryptophan hydroxylase (36). The second step leading to serotonin (5-hydroxytryptamine) is catalyzed by the cytoplasmic enzyme aromatic amino acid decarboxylase (37). This pyridoxal-dependent enzyme effects decarboxylation of L-dopa as well as 5-hydroxytryptophan in a constitutive fashion in the pineal. The conversion of serotonin to N-acetylserotonin, the immediate precursor of melatonin is highly regulated in the pineal and is catalyzed by serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) (FIG. 3) (38). Daytime levels of AANAT are up to 100-fold lower than nighttime levels in mammalian pineal glands, and it is this enzyme which is essentially fully responsible for the nighttime surge in melatonin production and drop in pineal serotonin (FIG. 4) (39, 40). The enzyme is acetyl-CoA dependent, and unlike the other enzymes in the pathway, had eluded characterization until very recently because of its instability and low abundance (41, 42). The final enzyme in melatonin biosynthesis is hydroxyindole-O-methyltransferase (HIOMT), a constitutive enzyme present in the pineal. HIOMT utilizes S-adenosylmethionine to effect O-methylation of the 5-hydroxy function of N-acetylserotonin (43). This enzymatic step appears to be minimally regulated and in most circumstances is not rate-limiting for melatonin production (5, 40).
AANAT activity was discovered in the early 1960's by Julius Axelrod (44). In 1970, Klein and Weller showed that this enzyme activity was regulated in a day/night cycle in the pineal gland and was primarily responsible for the day/night rhythm of melatonin (FIG. 4) (39, 40). Later, the enzyme was shown to be different from arylamine N-acetyltransferase, which is also present in the pineal (38). Because of its low abundance and instability, few mechanistic studies on the enzyme have been reported. Unlike the other enzymes involved in melatonin biosynthesis, no potent or specific inhibitors of AANAT have been reported.
In 1995, AANAT was cloned after many years of effort (41, 42). It is a 24 kDa (207 amino acids in length) protein with amino acid sequence highly conserved from chicken to man, showing&gt;80% amino acid identity between species (45, 46). Although AANAT shows little homology to other genes in the data bank, it does contain 2 short C-terminal regions (ca. 10-20 aa each) called motifs A (amino acids 121-140) and B (amino acids 165-177) which mark AANAT as a member of a superfamily of proteins (&gt;150 members so far identified, E. Koonin, NIH, private communication) that are believed to be largely composed of acetyl-CoA dependent transferase (45). Included in this family appear to be at least one member (PCAF) of the histone acetyltransferases (HATs) involved in the regulation of gene transcription (50). Very little is known about the substrate selectivity, mechanism, or inhibition of this enzyme family.